The herpes simplex virus (HSV) capsid plays a critical role in multiple steps of the virus life cycle. Early steps in infection include: fusion of viral and host membranes and release of the capsid into the cytoplasm; dynein- dependent trafficking of the capsid along microtubules to the nuclear envelope; docking of the capsid at a nuclear pore; and release of the viral genome into the nucleus. Binding of the capsid to the nuclear pore complex (NPC) appears to be mediated by the capsid protein pUL25 and the capsid-tethered tegument protein pUL36 through their interaction with the NPC proteins Nup214 and Nup358. In addition, NPC binding requires importin beta and a functional RanGTP/GDP cycle. A key knowledge gap in the early stages of infection is how the capsid engages the NPC and what triggers release of the viral genome from the capsid. The packaged HSV DNA creates a pressure of tens of atmospheres within the capsid and this pressure likely drives the translocation of the viral genome into the nucleus. Temperature-sensitive mutants in pUL25 and pUL36 have been found to dock at the NPC but both mutants fail to release DNA indicating that the role of these two proteins in NPC docking and uncoating can be separated. The objective of this proposal is to understand what triggers release of the packaged genome after docking of the capsid at the NPC. We have recently reported high-quality cryoEM reconstructions of the herpesvirus capsid imaged inside intact virions which allowed for a detailed model of subunit and domain organization of pUL25 and identified subunit contacts that pUL25 makes with capsid and with pUL36. Based on our preliminary studies of a novel pUL25 HSV mutant whose capsid binds the nuclear membrane but does not uncoat, we hypothesize that pUL25 initiates DNA release through its interaction with pUL36 and/or with one of the NPC proteins. We propose to test our hypothesis in two specific aims that i) define the role of pUL25 in capsid binding to the NPC and its role in triggering DNA release from the capsid; and ii) determine the structural changes in the capsid/NPC interaction that trigger HSV genome uncoating. These studies will examine the biochemical and structural basis of genome uncoating, provide unique information on how the capsid structure controls interactions with the NPC, and may reveal vulnerable structural intermediates as targets for blocking virus replication.